Self assembling proteins for producing extended materials

ABSTRACT

Self-assembling fusion proteins and nucleic acids encoding the same are provided. The subject fusion proteins include a first dimer forming oligomerization domain and a second tetramer forming oligomerization domain rigidly linked to each other. Also provided are regular structures made up of a plurality of self-assembled fusion proteins of the subject invention, and methods for producing the same. The subject fusion proteins find use in the preparation of self-assembled nanostructures, e.g., two-dimensional layers and three-dimensional networks, which structures find use in a variety of different applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.09/564,710 filed May 3, 2000; which application claims priority to thefiling date of U.S. Provisional Patent Application Ser. No. 60/133,470filed May 10, 1999; the disclosures of which applications are hereinincorporated by reference.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant No. GM31299awarded by the National Institute of Health; Grant No. MCB-0103549 fromthe National Science Foundation and Grant No. DE-FG03-87ER60615 awaredby the Department of Energy. The Government has certain rights in thisinvention.

INTRODUCTION

1. Technical Field

The field of this invention is nanotechnology and biomaterials.

2. Background of the Invention

The emerging field of nanotechnology has allowed the ability to designand fabricate novel small materials with sizes or length scales in thenanometer range that can serve complex functions. These materials fallinto a variety of architectural classes, such as compact clusters,hollow shells, tubes, two-dimensional layers, and three-dimensionalmolecular networks. These materials can subsequently be manipulated inreproducible ways to develop structures that have particular propertiesfor novel applications. For example, such applications include the useof such structures in the development of biological coatings, such as inprotein and DNA microarrays. Such nanotechnology materials have alsofound particular use in applications such as sensors and detectors, andas molecular sieves for filtration.

However, in view of recent developments in the filed of nanotechnologythere still remains a continued need and interest in the development ofnew materials and systematic methods for producing nanostructures usingsuch materials, especially for the development of biologicalmacromolecules for use in such applications. The present inventionaddresses this need.

RELEVANT LITERATURE

U.S. patents of interest include: U.S. Pat. No. 5,877,279; and U.S. Pat.No. 5,712,366. Articles of interest are: Collier, et al., Ann. Rev.Phys. Chem. (1998) 49: 371-404 (compact clusters); Rao, et al., CurrentOpinion in Solid State and Materials Sci. (1996) 1:279-284 and Kroto,Nature (1987) 329:529 (hollow shells); Iijima, Nature (1991)354:56-58,Ghadiri, Nature (1993)366:324-327 and Ajayan et al., Reports on Progressin Physics (1997) 60:1025-1062 (tubes); Stange, et al., Biophys. Chem.(1998) 72:73-85 (molecular networks); and Li, et al., Science (1999)283: 1145-1147; Seeman, Trends in Biotechnology (1999)) 11:437-443(DNA); and Chui, et al., Science (1999) 283:1148-1150 (two-dimensionallayers). Also of interest are: Padilla et al., PNAS (2001) 98(5)2217-21;Dotan et al., Angew. Chem. Int. Ed. Engl. (1999) 38(16):2363-2366;Winfree et al., (1998) 394(6693):539-544; Wukowitz et al., NatureStruct. Biol. (1995) 2:1062-1067; Ringler and Schulz. Science (2003)302(5642) 106-109.

SUMMARY OF THE INVENTION

Self-assembling fusion proteins and nucleic acids encoding the same areprovided. The subject fusion proteins include a first dimer formingoligomerization domain and a second tetramer forming oligomerizationdomain rigidly linked to each other. Also provided are regularstructures made up of a plurality of self-assembled fusion proteins ofthe subject invention, and methods for producing the same. The subjectfusion proteins find use in the preparation of self-assemblednanostructures, e.g., two-dimensional layers and three-dimensionalnetworks, which structures find use in a variety of differentapplications.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic illustration of a two-dimensional layer producedaccording to the present invention. Circles represent dimeric subunitsand squares represent tetrameric subunits. The arrows indicate the2-fold axis of the dimer units. The 4-fold axes of the tetramer come outof the page and go into the page for the black and grey squares,respectively. A single fusion protein (circled) consists of one subunitof the dimer and one subunit of the tetramer. The assembly extendsindefinitely to fill the plane.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Self-assembling fusion proteins and nucleic acids encoding the same areprovided. The subject fusion proteins include a first dimer formingoligomerization domain and a second tetramer forming oligomerizationdomain rigidly linked to each other. Also provided are regularstructures made up of a plurality of self-assembled fusion proteins ofthe subject invention, and methods for producing the same. The subjectfusion proteins find use in the preparation of self-assemblednanostructures, e.g., two-dimensional layers, which structures find usein a variety of different applications.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referenceunless the context clearly dictates otherwise. Unless defined otherwiseall technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit, unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and such embodiments are also encompassed within theinvention, subject to any specifically excluded limit in the statedrange. Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe invention.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing components that aredescribed in the publications that might be used in connection with thepresently described invention.

In further describing the subject invention, the subject fusionproteins, nucleic acids encoding the same and methods for producing thesame are described first in greater detail, followed by a review ofrepresentative structures (as well as specifically applications in whichthe same find use) which may be produced from the subject fusionproteins.

Fusion Proteins

As summarized above, the subject invention provides fusion proteins thatare capable of assembling under suitable conditions to produce regularstructures, e.g., two-dimensional layers. The fusion proteins of thesubject invention are characterized by having first and secondoligomerization domains joined together, e.g., covalently linked orfused together, through a linking group, such as a rigid linking group.The fusion proteins of the present invention may vary in size, dependingon the nature of the first and second oligomerization domains and anylinker present therein. In general, the subject fusion proteins mayrange in length from about 5° to about 1000, including from about 75 toabout 750 such as from about 100 to about 500 aa, and have a molecularweight ranging from about 5 kDa to about 100 kDA, such as from about 8kDa to about 80 kDa, including from about 10 kDa to about 50 kDa.

The oligomerization domains may have amino acid sequences that are foundin naturally occurring proteins, or may have sequences that arederivatives of sequences found in naturally occurring proteins, e.g.,where the domains are mutants of naturally occurring domains (includingpoint mutants, deletion mutants, substitution mutants, etc.).Alternatively, the oligomerization domains of the subject fusionproteins may have sequences that are not found in naturally occurringproteins, but instead are entirely synthetic. In many embodiments,however, the oligomerization domains have amino acid sequences that arefound in or derived from naturally occurring proteins. By naturallyoccurring protein is meant a protein that occurs in nature.

The oligomerization domains or components of the subject fusion proteinsare domains of stretches of amino acids that, under appropriateself-assembly conditions, such as physilogical conditions, associatewith one or or more identical domains to produce a stable, multimericstructure, e.g., a dimeric structure, a tetrameric structure. While thelength of a given oligomerization domain may vary, in many embodimentsthe length ranges from about 20 to about 500 aa, such as from about 50to about 400 aa, including from about 80 to about 300 aa. Accordingly,in many embodiments the weight of each oligomerization domain of thesubject fusion proteins may vary, but may range from about 2 to about 50kDa, such as from about 5 to about 40 kDa, including from about 8 toabout 30 kDa.

Generally, the oligomerization domains of the subject fusion proteinsare either: (a) a domain or stretch of amino acids which naturallyassociates into a dimeric structure (e.g., it is found in a protein thatassociates with an identical protein to produce a dimer); or (b) adomain or stretch of amino acids which naturally associates into atetrameric structure (e.g., it is found in a protein that associateswith three identical proteins to produce a tetramer). A furthercharacterization of the subject fusion proteins is that they include twodifferent oligomerization domains, i.e., a first oligomerization domainand a second oligomerization domain, where the first oligomerizationdomain is a domain that dimerizes with like domains, such that it is adimerization domain, and the second oligomerization domain is a domainthat associates into tetramers with three other identical domains, suchthat it is a tetramerization domain. Specific proteins of interest withknown three-dimensional structures that naturally associate intooligomeric (e.g., dimeric or tetrameric) structures include those dimersand tetramers listed in the publicly available Protein Data Bank,described in Abola et al., Meth. Enzymol. (1997) 277:556-571, and thelike.

The dimerization domains of the subject fusion proteins may have anyconvenient sequence of amino acids, so long as the sequence is such thatthe domain dimerizes with like domains, as described above.Representative dimerization domains of interest include, but are notlimited to: orange carotenoid protein from A. maxima (having amino acidsequences found at Genbank accession 28373616); M1 matrix protein frominfluenza virus (having amino acid and encoding nucleic acid sequencesfound at Genbank accession no. 3793307); and the like.

The tetramerization domains of the subject fusion proteins may have anyconvenient sequence of amino acids, so long as the sequence is such thatthe domain tetramerizes with like domains, as described above.Representative tetramerization domains of interest include, but are notlimited to: neuraminidase from influenza virus (having amino acid andencoding nucleic acid sequences found at Genbank accession no. 37785300;E. coli fuculose aldolase (having amino acid and encoding nucleic acidsequences found at Genbank accession no. 16130707); and the like.

A feature of the subject fusion proteins is that the two or morenaturally occurring protein components are joined to each other in asufficiently rigid manner such that the orientation in space of eachcomponent relative to the other(s) in the fusion protein is relativelystatic and can be anticipated in advance based on the known structuresof the components. Typically, the protein components of the subjectfusion proteins are joined to each other through a rigid linking groupthat is capable of providing the requisite static orientation of thedisparate components of the fusion protein. The length of the rigidlinking group may vary depending on the desired overall geometry of thefusion protein, as described below. Generally, the linking group has alength ranging from about 1.5 Å to 48 Å, such as from about 6 Å to 30 Å,including from about 6 Å to 20 Å. As such, the number of residues in thelinking group generally ranges from about 1 to 35, such as from about 2to 20, including from about 4 to 15.

Any linking group capable of providing the requisite static orientationof the disparate components of the fusion protein may be employed. Assuch, the linking group may hold the disparate components longitudinalto each other, such that the fusion proteins are aligned along the sameaxis, or the linking group may hold the components at an angle to eachother, e.g., at a right angle, such that axis of the firstoligomerization domain is perpendicular to the axis of the secondoligomerization domain. Of particular interest in certain embodiments isthe use of a linking group that includes an alpha helical structure. Inother words, the linking group may include a sequence of amino acidresidues that is prone to forming an alpha helix. A variety of suchsequences are known and include long alpha helices found in the proteinstructure database such as the helix in the ribosomal protein L9 (PDBcode 1div). Alternatively, it is understood that certain amino acidtypes tend strongly to adopt an alpha helical configuration, and thelinker may be designed to contain amino acids with this tendency. Inother embodiments, the linkage between the oligomerization domains maybe achieved by chemical bonds between amino acid side chains

A feature of the subject fusion proteins is that they are capable ofparticipating in a self-assembly process under suitable conditions toproduce a regular, defined structure of a plurality of fusion proteins.By plurality of fusion proteins is meant at least about 2, but thenumber of individual fusion proteins in a particular structure may bemuch higher, e.g., 5, 10, 15, 20, 30, 50, 100 or more, and sometimes avery large number, particularly in essentially infinitely repeatingstructures. As mentioned above, the subject fusion proteins are capableof self-assembling under suitable conditions, i.e., self-assemblyconditions, to produce regular structures. Suitable conditions are thoseconditions sufficient to provide for the self-assembly or association ofthe disparate fusion proteins into a regular structure. Representativeconditions under which self-assembly of the subject fusion proteinsoccurs are physiologic conditions or other laboratory conditions underwhich the individual component proteins are stable. By physiologicconditions is meant conditions found in living cell, e.g. a microbial,plant or animal cell. Typically, the conditions include an aqueousmedium having a pH ranging from about 4 to 10 and usually from about 6to 8, where the temperature ranges from about 4° C. to 35° C. However,it is understood that some proteins such as those from thermophilicmicroorganisms are stable under very extreme conditions and thatstructures from such stable components may have applications under suchconditions. In many embodiments, the self-assembling proteins willself-assemble with each other without the assistance or aid of accessoryproteins, e.g., chaperones, etc.

The subject fusion proteins are ones that self-assemble into regularstructures. By “regular structure” is meant that the structure has adefined pattern of assembly in two-dimensional or three-dimensionalspace that is known. In many embodiments, the subject fusion proteinsself-assemble into effectively infinitely repeating regular structures,such as two-dimensional layers. The subject fusion proteins assembleinto such regular structures because each oligomerization domain, e.g.,dimerization domain, tetramerization domain naturally occurring proteincomponent, serves as an oligomerization domain which provides for theassociation of the fusion proteins into the regular structure. As such,the relative orientations of the disparate components of the fusionprotein are selected to provide for the desired regular structure uponself-assembly under suitable conditions. Accordingly, for any givenfusion protein, the relative orientation of each component thereof ischosen based on the structure into which the fusion protein is designedto self-assemble. More specifically, the geometric relationship of thesymmetry elements of the oligomerization domains of the subject fusionproteins are chosen based on the desired regular structure.

The symmetry elements, i.e., symmetry axes, of a given fusion proteinare configured relative to each other, i.e., have a geometry, in amanner to provide for the overall symmetry required to produce thedesired structure. In many embodiments, the geometry of symmetryelements is non-intersecting. The geometry of the symmetries of each ofthe components is such that they are either parallel andnon-intersecting, or perpendicular and non-intersecting. In certainembodiments, the non-intersecting symmetry elements or axes form anangle that ranges from about 10 to about 90°, such as from about 25 toabout 75°, including from about 33 to about 66°, e.g., 45°, 50°, 55°,etc. The desired geometry may be achieved either by the design featuresof the part of the polypeptide chain that joins the two components, orby another covalent connection between the two components, e.g., adisulfide bond or the like.

Nucleic Acids Encoding the Fusion Proteins

Also provided by the subject invention are nucleic acid compositions. Bynucleic acid composition is meant a composition comprising a sequence ofnucleotides having an open reading frame that encodes a fusion proteinof the subject invention, as described supra. As such, the subjectnucleic acid compositions at least include a nucleic acid sequence thatencodes each of the oligomerization domains, where these sequences aregenerally joined by a sequence that encodes an amino acid sequence thatis prone to form an alpha-helical configuration. Though the length ofthe subject nucleic acid compositions may vary greatly depending on theparticular fusion protein that is encoded thereby, generally the subjectnucleic acid compositions are at least about 60 bp long, such as atleast about 150 bp long, including at least about 300 bp long, where thesubject nucleic acid compositions may be as long as 3 kbp or longer, butwill usually not exceed about 2 kbp in length.

The subject nucleic acid compositions may be produced by standardmethods of restriction enzyme cleavage, ligation and molecular cloning.One protocol for constructing the subject nucleic acid compositionsincludes the following steps. First, purified nucleic acid fragmentscontaining desired component nucleotide sequences as well as extraneoussequences are cleaved with restriction endonucleases from initialsources, e.g. animal cell, plant cell or microbial or viral genomes.Fragments containing the desired nucleotide sequences are then separatedfrom unwanted fragments of different size using conventional separationmethods, e.g., by agarose gel electrophoresis. The desired fragments areexcised from the gel and ligated together in the appropriateconfiguration so that a circular nucleic acid or plasmid containing thedesired sequences, e.g. sequences corresponding to the various elementsof the subject nucleic acid compositions, as described above, isproduced. Where desired, the circular molecules so constructed are thenamplified in a prokaryotic host, e.g. E. coli. The procedures ofcleavage, plasmid construction, cell transformation and plasmidproduction involved in these steps are well known to one skilled in theart and the enzymes required for restriction and ligation are availablecommercially. (See, for example, R. Wu, Ed., Methods in Enzymology, Vol.68, Academic Press, N.Y. (1979); T. Maniatis, E. F. Fritsch and J.Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1982); Catalog 1982-83, NewEngland Biolabs, Inc.; Catalog 1982-83, Bethesda Research Laboratories,Inc.

The above nucleic acid compositions find use in the preparation of thesubject fusion proteins.

Methods of Preparing the Subject Fusion Proteins

The subject fusion proteins are obtained by expressing a recombinantgene encoding the fusion proteins, such as the polynucleotidecompositions described above, in a suitable host. For expression, anexpression cassette may be employed. The expression vector will providea transcriptional and translational initiation region, which may beinducible or constitutive, where the coding region is operably linkedunder the transcriptional control of the transcriptional initiationregion, and a transcriptional and translational termination region.These control regions may be derived from a variety of sources.

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Expression cassettes may beprepared comprising a transcription initiation region, the regionencoding the fusion protein, and a transcriptional termination region.After introduction of the DNA, the cells containing the construct may beselected by means of a selectable marker, the cells expanded and thenused for expression. The expression cassette contained in the cell maybe as part of an extrachromosomal element or integrated into the genomeof the cell as a result of introducing the expression cassette into thecell. Accordingly, in many embodiments, the expression cassette will bemaintained in the host cell and passed on to cellular progeny of thehost cell.

The proteins may be expressed in prokaryotes or eukaryotes in accordancewith conventional ways, depending upon the purpose for expression. Forlarge scale production of the protein, a unicellular organism, such asE. coli B. subtilis, S. cerevisiae, insect cells in combination withbaculovirus vectors, or cells of a higher organism such as vertebrates,particularly mammals, e.g. COS 7 cells, may be used as the expressionhost cells. In some situations, it is desirable to express the proteinsin eukaryotic cells, where the encoded protein will benefit from nativefolding and post-translational modifications.

Where desired, the protein may be purified following its expression toproduce a purified protein comprising composition. Any convenientprotein purification procedures may be employed, where suitable proteinpurification methodologies are described in Guide to ProteinPurification, (Deuthser ed.) (Academic Press, 1990). For example, alysate may be prepared from the original source, e.g. the expressionhost expressing the protein, and purified using HPLC, exclusionchromatography, gel electrophoresis, affinity chromatography, and thelike.

Preparation of Regular Structures

The subject fusion proteins find use in the production of various typesof regular structures, i.e., structures of defined and predictablegeometry. Specifically, the subject fusion proteins find use in thepreparation of nanosized two-dimensional crystalline layers orthree-dimensional crystalline networks, where such layers or networksmay in theory be of infinite size (e.g., length and width) but in manyembodiments range in linear dimension from about 20 nm to about 1 mm,such as from about 50 nm to about 500 um, including from about 100 nm toabout 200 μm,

To prepare regular structures from the subject fusion proteins, thefusion proteins are generally combined under conditions sufficient forself-assembly of the fusion proteins into the desired regular structureto occur. Representative conditions that promote self-assembly arephysiologic conditions, as mentioned above. The concentration of thefusion protein in the medium must be sufficiently high such thatself-assembly into the desired structure occurs. Typically, the fusionprotein concentration is at least about 0.05 mg/ml and more usually atleast about 0.25 mg/ml.

In many embodiments, the structures are assembled from a plurality ofidentical fusion proteins, i.e., they are homogenous with respect to thefusion protein. In such embodiments, preparation of the fusion protein(e.g. expression of a nucleic acid encoding the protein) may occur inthe same reaction medium as assembly of the structure, e.g., in the hostcell used to express the fusion protein. Alternatively, in otherembodiments, preparation of distinct fusion proteins (e.g., expressionof nucleic acids encoding the proteins) may occur in separate media,prior to assembly of the heterogeneous structure.

Utility

The regular structures produced by the self-assembly of the subjectfusion proteins find use in a variety of different applications. Asmentioned above, structures can be assembled that resembletwo-dimensional layers. Such ordered two-dimensional protein layers finduse as biological coatings, sensors, detectors, molecular sieves,substrates for the attachment of specific chemical groups at ultra-highdensity, as porous materials for filtration with precise pore sizes onthe mid-nanometer scale, and the like. Likewise, three-dimensionalnetwork materials may find use as sensors, porous catalysts, materialsfor slow release of encapsulated compounds, and the like.

Kits

Also provided are kits for use in producing the subject fusion proteinsand self-assembled regular structures. The subject kits at least includea nucleic acid composition that encodes a fusion protein, where thenucleic acid is typically present on a vector. The kits may furtherinclude expression hosts suitable for expressing the subject fusionproteins. Also provided in the kits may be other reagents useful forproducing the subject fusion proteins, e.g. buffers, growth mediums,enzymes, selection reagents, and the like.

In addition to above-mentioned components, the subject kits typicallyfurther include instructions for using the components of the kit topractice the subject methods. The instructions for practicing thesubject methods are generally recorded on a suitable recording medium.For example, the instructions may be printed on a substrate, such aspaper or plastic, etc. As such, the instructions may be present in thekits as a package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging or subpackaging)etc. In other embodiments, the instructions are present as an electronicstorage data file present on a suitable computer readable storagemedium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actualinstructions are not present in the kit, but means for obtaining theinstructions from a remote source, e.g. via the internet, are provided.An example of this embodiment is a kit that includes a web address wherethe instructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental

In order to demonstrate a two-dimensional protein layer, self-assemblinginto a square, repeating pattern, we fused a cyclic tetrameric protein,fuculose aldolase from E. coli, to a dimeric protein, M1 matrix proteinfrom influenza virus. These two oligomerization domains were connectedin the larger fusion protein by a short flexible linker. Specifically,residues 1-206 of fuculose aldolase were fused to residues 1-187 of theM1 matrix protein via a 5-residue linker with amino acid sequence DPVPV.This fusion resulted in a 398 residue fusion protein with an N-terminalfuculose aldolase domain and a C-terminal M1 matrix protein domain,having a molecular weight of 41 kDa. The geometrically rigid connectionbetween the two domains was achieved by way of a covalent bond betweenindividual cysteine residues in the two oligomerization domains. Thesecysteine residues were genetically engineered into the two domains inadvance, specifically at residue 86 of fuculase aldolase and residue 34of M1 matrix protein. According to the geometry of the design, with thesymmetry axes of the two oligomerization domains being non-intersectingbut forming an angle very nearly equal to 90 degrees, the fusionproteins are designed to assemble into an extended two-dimensional layerwith a symmetry that would be described by the notation p42₁2.

A multistep PCR protocol was used to create the construct describedabove from the mutated dimer and tetramer clones. This construct wasligated into a pET-21b vector between the NdeI and EcoRI sites, adding ahistidine tag to the N-terminus, where it is not expected to interferewith the intramolecular disulfide formation or subsequent layerassembly. This vector was transformed into E. coli BL21-DE3 cells. Thecells were grown at 37° C. and induced with IPTG at an optical densityof 0.6. The protein is produced in milligram quantities, but is presentinside the host cells in inclusion bodies. Therefore, the protein waspurified from inclusion bodies in the presense of dithiothreitol andurea.

The resultant purified protein self assembles into a two-dimensionalprotein layer, having a square, repeating pattern.

It is evident from the above results and discussion the subjectinvention provides powerful tools and methodologies for producingordered structures from self-assembling fusion proteins. The fusionproteins of the subject invention can be readily produced and thenself-assembled into a variety of different structures which find use ina plurality of different applications. As such, the subject inventionrepresents a significant contribution to the field.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A fusion protein comprising a first oligomerization domain thatnaturally associates into homodimeric structures and a secondoligomerization domain that naturally associates into homotetramericstructures, wherein said first and second oligomerization domains arerigidly linked to each other.
 2. The fusion protein according to claim1, wherein said first and second oligomerization domains are derivedfrom naturally occurring proteins.
 3. The fusion protein according toclaim 1, wherein said first and second oligomerization domains arerigidly linked to each other by a linking group.
 4. The fusion proteinaccording to claim 1, wherein said first and second oligomerizationdomains have a geometry such that their symmetry axes arenon-intersecting.
 5. The fusion protein according to claim 4, whereinsaid first and second oligomerization domains have a geometry such thattheir symmetry axes are parallel.
 6. The fusion protein according toclaim 4, wherein said first and second oligomerization domains have ageometry such that their symmetry axes are non-intersecting andperpendicular.
 7. The fusion protein according to claim 4, wherein saidfirst and second oligomerization domains have a geometry such that theirsymmetry axes are non-intersecting and form an angle of 45 degrees.
 8. Afusion protein comprising a first oligomerization domain that naturallyassociates into homodimeric structures and a second oligomerizationdomain that naturally associates into homotetrameric structures linkedto each other by an alpha helical linking group.
 9. The fusion proteinaccording to claim 8, wherein said first and second oligomerizationdomains have a geometry such that their symmetry axes arenon-intersecting.
 10. The fusion protein according to claim 8, whereinsaid first and second oligomerization domains have a geometry such thattheir symmetry axes are parallel.
 11. The fusion protein according toclaim 8, wherein said first and second oligomerization domains have ageometry such that their symmetry axes are non-intersecting andperpendicular.
 12. The fusion protein according to claim 8, wherein saidfirst and second oligomerization domains have a geometry such that theirsymmetry axes are non-intersecting and form an angle of 45 degrees. 13.A regular structure produced by the self-assembly of a plurality offusion proteins according to claim
 1. 14. The regular structureaccording to claim 13, wherein said structure is homogenous with respectto its fusion protein components.
 15. The regular structure according toclaim 13, wherein said regular structure is a two-dimensional layer. 16.The regular structure according to claim 13, wherein said regularstructure is a three-dimensional crystalline network.
 17. A method ofproducing a regular structure, said method comprising: producing aplurality of fusion proteins according to claim 1; and combining saidplurality of fusion proteins under conditions sufficient for saidregular structure to form.
 18. The method according to claim 17, whereinsaid conditions are physiologic conditions or other laboratoryconditions under which the component oligomerization domains would bestable.
 19. The method according to claim 17, wherein said producing andcombining steps occur in the same reaction medium.
 20. The methodaccording to claim 17, wherein said producing and combining steps occurin separate media.
 21. A nucleic acid encoding a fusion proteinaccording to claim
 1. 22. An expression cassette comprising atranscriptional initiation region functional in an expression host, anucleic acid having a nucleotide sequence found in the nucleic acidaccording to claim 21 under the transcriptional regulation of saidtranscriptional initiation region, and a transcriptional terminationregion functional in said expression host.
 23. A cell comprising anexpression cassette according to claim 22 as part of an extrachromosomalelement or integrated into the genome of a host cell as a result ofintroduction of said expression cassette into said host cell.
 24. Thecellular progeny of the host cell according to claim
 23. 25. A kitcomprising a nucleic acid according to claim
 21. 26. A fusion protein ofat least two oligomerization domains rigidly linked to each other,wherein said fusion protein is capable of self-assembling withadditional fusion proteins to produce a regular structure.